JOURNAL OF CLINICAL MICROBIOLOGY, May 1994, p. 1268-1272 Vol. 32, No. 50095-1 137/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Single-Tube, Noninterrupted Reverse Transcription-PCR forDetection of Infectious Bursal Disease Virus

LONG HUW LEE,* LU JEN TING,t JUI HUANG SHIEN, AND HAPPY K. SHIEHDepartment of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan 403

Received 15 November 1993/Returned for modification 5 January 1994/Accepted 15 February 1994

An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for thedetection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had beenoptimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas nonewas obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCRwas tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidiumbromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens fromcommercially reared chickens. The identity of the amplified products from the tissue specimen preparation wasdetermined by using a rapid, simple procedure in which internally nested, end-labeled probes were used.

Infectious bursal disease virus (IBDV) infections cause avariety of disease syndromes in young chickens. These rangefrom loss of feeding efficiency (13) to ablation of the antibodyimmune response (25). At least two serotypes of the virus arecurrently recognized (5, 14); the known serotype 1 viruses arepathogenic only in chickens, and pathogenic serotype 2 viruseshave not been isolated (2). During the past few years, severalworkers have described new virulent chicken isolates whichrepresent either a different serotype or variants of the classicstrains isolated before 1985 and currently circulating in thefield (18, 19). IBDV is classified as a birnavirus (8). The virusgenome consists of two segmented double-stranded RNAs(dsRNAs) (16). The larger segment (segment A) is approxi-mately 3.4 kbp in length, while the smaller segment (segmentB) is approximately 2.9 kbp long (1).The methods for diagnosis of IBDV infection include agar

gel precipitation, virus neutralization, and enzyme-linked im-munosorbent assay (ELISA). They are designed to measurelevels of antibodies to IBDV. In addition, direct immunofluo-rescence for demonstrating the presence of viral antigen hasbeen routinely used with tissue sections or impression smears(15). Antigen capture ELISA has been described for thedetection of IBDV antigens directly from the infected bursaltissues (9, 23). Radiolabeled (6) and nonradiolabeled cDNAprobes (7, 9a) used for detecting IBDV RNA have recentlybeen considered more sensitive than the other methods.Lately, another method, reverse transcription-PCR (RT-PCR), has been described for the diagnosis of IBDV infection(12). The sensitivity of this technique could be further en-hanced by hybridization of amplification products after South-ern transfer. Attempts have been made to minimize thenumber of manual manipulations required for processing alarge number of samples. Recently, a system was designedwhereby all the reagents required for both RT and PCR can beadded to a single tube and a simple, noninterrupted thermalcycling program can be carried out for detection of rotavirus(26) and Ross River virus (21). Here we describe a combined

* Corresponding author. Mailing address: Department of Veteri-nary Medicine, National Chung Hsing University, Taichung, Taiwan403. Phone: (04) 286-0196. Fax: (04) 286-0196.

t Present address: Taiwan Provincial Research Institute for AnimalHealth, Tansui, Taipei, Taiwan.

RT-PCR assay for the detection of IBDV. The identity of PCRamplification products was determined by mixing the productswith an internally nested, end-labeled oligonucleotide probefollowed by one cycle of PCR.

MATERIALS AND METHODS

Viruses and cells. Three strains of IBDV representing bothserotypes 1 and 2 were used. MO is a serotype 2 turkey isolate(11), while P3009 and 29/11 are isolates locally obtained fromchickens. They have been described as serotype 1 viruses (10).All viruses were propagated in chicken embryo fibroblasts(CEF) as described by Lee and Lukert (11). Seven unrelatedavian pathogens were used to determine the specificity of theoligonucleotide primers. The six viruses tested were the com-mercially available vaccine strains and included infectiousbronchitis virus (Intervet International B. V., Boxmeer, Hol-land), turkey herpesvirus and Newcastle disease virus (SalsburyLaboratories), avian reovirus strain 1133 (Vineland Laborato-ries, Vineland, N.J.), and infectious laryngotracheitis virus andfowlpox virus (Nippon Institute for Biological Science, Tokyo,Japan). In addition, an avian influenza virus (20) was also usedfor the specificity test.

Viral RNA purification. Virus particles of IBDV strainsP3009, 29/11, and MO were separately purified by methodsdescribed previously (9). Briefly, after the viruses had beenpropagated in CEF and concentrated by polyethylene glycol6000 they were pelleted and resuspended in TNE buffer (0.01M Tris-HCl, pH 7.6; 0.1 M NaCl; and 0.001 M EDTA),followed by extraction with Freon TF (DuPont, Sydney, Aus-tralia). The aqueous phase was cushioned through 35% su-crose, and the virus-containing pellets obtained were thencentrifuged isopycnically in stepwise gradients of 40, 30, and20% CsCl2. The viruses banding at a buoyant density of 1.33g/ml in CsCl2 gradient were withdrawn and were pelleted bycentrifugation at 132,000 x g at 4°C for 2 h. The pellets wereresuspended in TNE buffer containing 0.5% sodium dodecylsulfate, and proteinase K (Boehringer Mannheim) was addedto a final concentration of 1 mg/ml. After incubation at 37°Cfor 2 h, the viral RNA was extracted with phenol and chloro-form according to the standard procedures. Viral dsRNA wasthen purified by differential LiCl precipitation (3) and washedtwice with 70% ethanol to remove LiCl. Finally, purified viral

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IBDV DETECTION BY ONE-TUBE, NONINTERRUPTED RT-PCR 1269

IDBV g,nome@ *-egn-nt A ( 3411 bp )

a IKbp

VP

313 bp

VPJ

VP'

FIG. 1. IBDV genome segment A and PCR fragment. The blackbar represents genome segment A of IBDV strain Cu-1. The respectiveregions of the genome encoding viral proteins are indicated byunshaded bars. The segment of VP2 marked 318 bp displays thefragment size amplified by PCR. The arrow indicates the direction andsize of the internally nested, end-labeled probe.

dsRNA was resuspended in TE buffer (0.01 M Tris-HCI, pH8.0; 0.001 M EDTA) until used.

Preparation of nucleic acid from tissue specimens. Bursal-tissue specimens (specimens 1 to 23) were collected from 21different commercial broiler chicken farms where IBDV infec-tions were suspected. Specimens 2 and 3 have been previouslyexamined with monoclonal antibody probes to IBDV strainP3009 by immunodot assay (9) and RT-PCR (12). Specimen 2(designated specimen J in previous reports) was identified as

IBDV negative, whereas specimen 3 (designated specimen Gin previous reports) was IBDV positive. Each sample to betested contained a pool of two to four bursae, which were

homogenized in TNE buffer (3 ml per bursa). Followinglow-speed centrifugation, a 50-,ul volume of the original bursalhomogenate was mixed with 1 ml of lysis buffer (8 M guani-dinium HCl, 0.1 M EDTA, 0.3 M sodium acetate) for 20 minat 0°C and then centrifuged at 12,000 x g for 20 min at 4°C.The supernatant was collected, and nucleic acids were precip-itated with ethanol.

Preparation of nucleic acid from unrelated avian pathogens.The specificity of the primer set for IBDV was further deter-mined by using the IBDV strains (P3009, 29/11, and MO) andthe seven unrelated avian viruses mentioned above. Thenucleic acids were directly extracted from a minimum of 1,000infectious virions. The lyophilized viruses were resuspended inTNE buffer. Nucleic acids were released by treatment with lysisbuffer and were precipitated with ethanol as described forbursal-tissue specimens.

Oligonucleotide primers for RT-PCR and the internallynested, end-labeled probe. Primers were chosen according tothe cDNA sequence of the IBDV strain Cu-I genome seg-ments reported by Spies et al. (24). Primers were selected inthe part of the IBDV genome segment A coding for VP2capsid protein (Fig. 1). The sequences of primers for RT-PCRare as follows: pl, 5'-GAGATCAGACAAACGGGATCGCA-3' (identical to nucleotides 72 to 92, numbered accordingto reference 24); p2, 5'-GTAGYTGTAACTGGCCGG-3'(complementary to nucleotides 372 to 389). The sequence ofthe oligomer used as the internally nested, end-labeled probeis identical to nucleotides 249 to 263.RT-PCR. The system was designed so that all components

for both RT and PCR could be combined in one 0.5-mlEppendorf tube and both reactions could be run in a simplenoninterrupted thermal cycling program. Standard conditionsfor RT of the viral RNA and amplification of the 318-bpsequence were as follows. Each reaction tube contained targetviral RNA at various dilutions; 0.5 p.M each primer; 100 p.Meach the four deoxynucleoside triphosphates; 1.5 mM MgCl2;1 x reaction buffer containing 10 mM Tris-HCI (pH 8.0), 50

mM KCl, 1.5 mM MgCI2, and 0.1 mg of gelatin per ml; 2 U ofSuperscriptase (a cloned RNase H free reverse transcriptase)(Bethesda Research Laboratories); and 2.5 U of Taq DNApolymerase (Boehringer Mannheim) in a total volume of 20 [Ll.The mixture was boiled for 5 min to denature the target RNAand cooled on ice for 2 min before the two enzymes wereadded. Each reaction mixture was covered with 20 pL1 ofmineral oil (Sigma Chemical Company) and incubated in aPerkin-Elmer Cetus DNA thermal cycler according to thefollowing protocol: 40 min at 45°C (to allow RT) and 5 min at95°C (for inactivation of Superscriptase and denaturation ofDNA); 1(0 cycles of 94°C for 45 s, 55°C for 35 s (primerannealing), and 72°C for 1 min (primer extension); 45 cycles of91°C for 45 s, 55°C for 35 s, and 72°C for 40 s; and then a finalincubation for 10 min at 72°C. Conditions that were subse-quently altered were the concentrations of Superscriptase, Taqpolymerase, and MgCl2 and the number of PCR cycles.

Analysis of the PCR product. A 5-,u volume of the PCRproducts was separated on a 2% agarose gel (100 V for 30 minin TBE buffer [89 mM Tris-HCl, 89 mM boric acid, 2 mMEDTA, pH 8.3]). DNA of plasmid pBR322 digested withHaeIII (Boehringer Mannheim) was electrophoresed as a sizemarker to determine the length of the amplified fragment. Analternative method for analyzing PCR products was essentiallyas described previously (17). Briefly, the reaction mixturecontained 8 ,ul of the PCR products, 0.2 pmol of the internallynested, R-[32P]ATP end-labeled (polynucleotide kinase;Boehringer Mannheim) oligomer probe, and 0.5 U of Taqpolymerase in a total volume of 10 ,ul. After addition of 20 p.1of mineral oil, the mixture was subjected to one cycle (dena-turation for 20 s at 98°C, annealing for 1 min at 50°C, extensionfor 3 min at 72°C) in a thermal cycler as described above. ThePCR products were then separated on a 2% agarose gel anddried for 40 min under a vacuum with a gel dryer and exposedto Kodak X-ray film.

RESULTS

RT-PCR conditions. To determine the optimum ratio of Taqpolymerase and Superscriptase for RT-PCR, reactions wereperformed with various proportions of the two enzymes.Firstly, six RT-PCRs were performed with 2 U of Super-scriptase and 0.5, 1.0, 1.5, 2.0, 2.5, and 3 U of Taq polymerase,respectively, with purified RNA of IBDV strain P3009 as thetarget sequence. All reactions were successful, showing moreproduct formation as Taq polymerase concentration increased,but the amount of nonspecific products slightly increasedsimultaneously (Fig. 2A). A set of five similar RT-PCRs wereperformed with 2.5 U of Taq polymerase and 100, 50, 10, 5, and2 U of Superscriptase, respectively. All these reactions werealso successful; however, the band brightness increased as theconcentration of Superscriptase decreased (Fig. 2B). There-fore, the concentrations of Taq polymerase and Superscriptasechosen for RT-PCR were 2.5 and 2 U, respectively. In addi-tion, five RT-PCRs were carried out to determine the optimumMgCl2 concentration. Each reaction mixture contained theoptimum ratio of the two enzymes, with the same purifiedRNA as the target sequence. MgCl2 at concentrations of 0, 1.0,1.5, 3.0, and 4.5 mM was added to each of the five reactionmixtures, respectively. The results indicated that all reactionsexcept for the one without addition of MgCl, were successful,and more products were formed as the MgCl2 concentrationincreased. However, nonspecific products were formed, andthe specific product was notably missing in the reaction withthe addition of 4.5 mM MgCl2. Because the RT-PCR bufferoriginally contained 1.5 mM MgCl,, the total MgCl, concen-

X -vp

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1270 LEE ET AL.

A bp M 1 2 3 4 5 6 7 8 9 1011 1213 14

bp M 1 2 3 4 5 6

- 318bp- 318bp

B

1 2 3 4 5

3l8bp

FIG. 2. Amplification of the fragment (318 bp) from dsRNA ofIBDV strain P3009. (A) Reaction mixes for lanes I to 6 contain 2 U ofSuperscriptase each and 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 U of Taqpolymerase, respectively. Lane M contains DNA of plasmid pBR322digested with HaeIII. (B) Reaction mixes for lanes 1 to 5 contain 2.5 Uof Taq polymerase each and 100, 50, 10, 5, and 2 U of Superscriptase,respectively.

tration chosen for RT-PCR was 3.0 mM. To determine theeffect of the number of PCR cycles on optimum productformation, reactions were conducted with totals of 40, 50, and60 cycles. The results showed that all reactions were successful,and more product was formed as the number of cyclesincreased. However, nonspecific products start to be observedat 60 reaction cycles. Thus, the number of cycles chosen forRT-PCR was 55.

Detection of RNA of IBDV strains. Purified RNA from CEFinfected with IBDV strains P3009, 29/11, and MO was used forRT-PCR, as described in Materials and Methods. Analysis ofthe amplified products in an agarose gel indicated the presenceof DNA bands of the expected size. Amplified DNA was notdetected when nucleic acids from mock-infected cells wereused. The identity of the amplified DNA fragments with thepublished sequence of the Cu-1 strain (24) was further verifiedby sequencing the amplified products by using a dsDNA cyclesequencing system (Bethesda Research Laboratories). Thus,the amplified DNA products from the three IBDV strains useddid not originate from contamination with unknown DNA butwere the products of the designated region.

Sensitivity and specificity of RT-PCR. RT-PCR of threeIBDV strains was carried out, and nucleic acids extracted frommock-infected CEF were included as a negative control. ViralRNA was added to RT-PCR mixtures in 10-fold dilutions from10 pg to 1 fg of RNA per reaction. After electrophoresis, it wasfound that RT-PCRs with 10 pg to 10 fg of IBDV RNAproduced the expected 318-bp product in an ethidium bro-mide-stained gel but those with 1 fg of IBDV RNA and nucleicacids from uninfected CEF did not produce any amplifiedproduct. To assess the specificity of detection of IBDV RNAby RT-PCR, seven unrelated poultry viruses which includedboth RNA and DNA viruses were tested by RT-PCR of theirnucleic acids. Three IBDV strains were used as a positivecontrol. No fragment could be amplified either with the nucleicacids extracted from any of the unrelated viruses tested or fromuninfected bursal tissue of chickens or with the cell lysis buffer;however, the amplified product of 318 bp was detected with thenucleic acids from the three IBDV strains.

Detection of fragments from bursal-tissue specimens. RT-

FIG. 3. Amplification of the 318-bp fragment from bursal-tissuespecimens (lanes 2 to 14). Lane 1 is a negative control containing celllysis buffer. Lane M contains a molecular size marker, as indicated inFig. 2. Arrows indicate the 318-bp amplified fragments.

PCR was carried out for detection of IBDV RNA from 23bursal-tissue specimens. An amplified product of 318 bp wasdetected from 19 of the specimens following ethidium bromidestaining. Amplified product was not obtained when the celllysis buffer was used as a negative control. Some of the resultsare indicated in Fig. 3. The identity of the amplificationproduct from the bursal-tissue specimens was further verifiedby one additional cycle of PCR with the internally nested,end-labeled oligomer probe. Partial results are indicated inFig. 4. Oligomer extension products were obtained from someof the samples that showed an amplification product at 318 bp.

DISCUSSION

RT-PCR has been developed for detecting IBDV RNA (12),but the protocols always require two separate major stages: RTand a PCR cycle. Following termination of RT-PCR, theidentity of the amplified product was usually determined bySouthern hybridization. The results of the present studiesindicated that RNA of IBDV can be successfully detected byusing a simplified RT-PCR protocol in which the two stagesare combined and run sequentially in a single tube withoutremoving the tube from the PCR machine. These proceduresare easier and faster than the early RT-PCR procedures (12),particularly when a large number of tissue specimens are

examined. Furthermore, the identity of the amplification prod-uct could be determined by an internally nested, end-labeledoligonucleotide probe with one cycle of PCR. However, oli-gomer extension products were not obtained from some spec-imens that showed an amplification product at 318 bp. Ingeneral, the signal appeared to be weaker with the oligonucle-otide probe (Fig. 4), because the volume of PCR product usedfor oligomer extension by one cycle of PCR is even more thanthat used with an ethidium bromide-stained gel (Fig. 3).Therefore, oligomer extension with one cycle of PCR for theidentification of PCR product may not be successful underoptimum conditions, leading to a decrease in sensitivity.The sensitivity of the primer set was assayed by using

different concentrations of purified viral RNA, and the limit ofdetection for viral RNA was 10 fg. In addition, this primer setappears to be specific for IBDV RNA, because it did notamplify the DNA sequences of nucleic acid preparations frommock-infected CEF and uninfected bursal tissue. Furthermore,the primer set did not amplify the DNA sequences of nucleicacid preparations from seven unrelated avian viruses tested. Inaddition to detecting two serotype 1 viruses, this primer setalso detects RNA from one strain of IBDV serotype 2 virus.

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IBDV DETECTION BY ONE-TUBE, NONINTERRUPTED RT-PCR 1271

1 2 3 4 5 6 7 8 9 1011 121314

E.

p

-N

FIG. 4. Determination of the identity of amplified products frombursal-tissue specimens (lanes 2 to 14). Lane 1 is a negative controlcontaining cell lysis buffer. P, oligomer extension products; N, nonhy-bridized probes.

This suggests that this primer set may also have broad speci-ficity for IBDV strains.A systematic investigation of the relationship between re-

verse transcriptase (RTase) and Taq polymerase activities hasbeen described (22). This study demonstrated that RTasesfrom both avian myeloblastosis virus and Moloney murineleukemia virus are able to block Taq polymerase activity if theRTase/Taq polymerase ratio is greater than approximately 3:2.Taq polymerase activity may be completely inhibited when theRTase concentration increases because the amplification prod-uct is not obtained. Hence, Sneller et al. (22) suggest that theoptimum RT-PCR conditions for the detection of viral RNAare 0.5 U of RTase and 2 U of Taq polymerase. Thiscombination allows combined RT and PCR in one reactiontube without interruption, while minimizing the inhibitoryeffect of RTase on Taq polymerase. The results obtained in thepresent study indicated that Superscriptase was able to blockTaq polymerase activity, similar to the results reported bySneller et al. (22) (Fig. 2). However, the maximum Super-scriptase/Taq polymerase ratio for inhibiting Taq polymeraseactivity reported here is different from the ratio reported bySneller et al. (22). When the Superscriptase/Taq polymeraseratio increases to 10:2.5, Taq polymerase activity seems not tobe affected. However, if the ratio increases to 50:2.5 or greater,the amplification activity then decreases significantly but is notcompletely stopped. Because the Superscriptase used in thisstudy is a cloned RNase H, free RTase originated fromMoloney murine leukemia virus. RNase H deletion from theRTase molecule may affect this blocking activity. Alternatively,the number of PCR cycles may also affect observation of theamplified product. When the same RT-PCR mixtures wereprepared and 35 cycles were run, the amount of productamplified from each reaction decreased, and no product wasobtained with reaction mixtures containing Superscriptase andTaq polymerase at ratios of 50:2.5 and 100:2.5 (data notshown).

Theoretically, the amount of amplification product increasesif PCR cycles are extended. However, this increase will be

limited by several factors. One of them *is the enzymaticstability of Taq polymerase following thermal cycle reactions.The half-lives of Taq polymerase are 130 and 40 min at 92.5and 95°C, respectively (4). Hence, if an attempt to increase theamount of amplification product (sensitivity) by increasing thenumber of PCR cycles was made, the temperature for dena-turation of the DNA template had to be considered. Theresults show that the 318-bp amplified product was obtainedand the product formation increased in parallel with thenumber of cycles after 10 cycles at 94°C followed by variousnumbers of cycles at 91°C for denaturation (Fig. 2B).

Thus, the results show that a sensitive and specific, single-tube RT-PCR seems to be a viable alternative tool for detect-ing IBDV.

ACKNOWLEDGMENT

This work was supported by the Council of Agriculture, Republic ofChina.

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